Tuberculosis represents a major health burden that requires more investigation to better understand the factors involved in the entry and establishment of chronic stage of the disease. The molecular pathogenesis of chronic tuberculosis (approximately 95% of infections), where mycobacterial bacilli stay in a phenotypically dormant state despite being metabolically active, is far from being understood (3). Previously, we utilized a technology developed by our group (In Vivo Microarrays Analysis, IVMA) to identify Mycobacterium tuberculosis (M. tb) genes responsible for entering into the chronic stage of tuberculosis. The IVMA analysis of chronic tuberculosis suggested a prominent role for rv0990c in establishing the chronic tuberculosis. Further microarrays analysis of an isogenic mutant of the rv0990c strain (?rv0990c) suggested that Rv0990c could regulate the expression of a large number of genes. Bioinformatics analysis of the rv0990c sequence indicated it encodes a member of a large family of proteins responsive to heat shock. Gene specific transcriptional profiling further confirmed that transcripts of rv0990c were induced following exposure to heat shock and long-term hypoxia. Following animal infections, the ?rv0990c strain was attenuated in BALB/c mice compared to its parent (H37Rv) or complemented (?rv0990c::rv0990c) strains. We hypothesize that the rv0990c gene product participates in regulating a large number of proteins involved in heat shock responses necessary for mycobacterial survival during infection. The main focus of this project is to explore the role and potential function(s) of rv0990c in the pathogenesis of M. tb. In the first aim, we will use RNA Sequence (RNASeq) profiling to identify stress responsive genes that are impacted by the rv0990c gene using 2 in vitro models for heat shock and multiple stressors. In the second aim, we will use antibody pull-down assays and yeast 2-hybrid like system to identify binding partners for Rv0990c during heat shock responses. Finally, we will monitor the survival and phagosome maturation of the rv0990c mutant in murine bone marrow-derived macrophages. We believe that the approaches exploited in this proposal will examine the role of a novel regulatory protein in M. tb pathogenesis. The proposed studies will further improve our understanding of the nature of the host-pathogen interactions and will provide the necessary foundation for more detailed and mechanistic studies on the role of rv0990c in M. tb virulence. PUBLIC HEALTH RELEVANCE: This project focuses on better understanding of the molecular basis of tuberculosis, a worldwide health problem to mankind. This project is highly relative to the mission of the NIH/NIAID.